EP1414478B1 - Hemmung der neurodegeneration - Google Patents

Hemmung der neurodegeneration Download PDF

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EP1414478B1
EP1414478B1 EP02739383A EP02739383A EP1414478B1 EP 1414478 B1 EP1414478 B1 EP 1414478B1 EP 02739383 A EP02739383 A EP 02739383A EP 02739383 A EP02739383 A EP 02739383A EP 1414478 B1 EP1414478 B1 EP 1414478B1
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ntp
ad7c
expression
gene
cells
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EP1414478A2 (de
EP1414478A4 (de
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Jack R. Wands
Suzanne M. De La Monte
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Rhode Island Hospital
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Rhode Island Hospital
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A01K2267/0312—Animal model for Alzheimer's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • This invention relates to neurodegenerative conditions.
  • Alzheimer's Disease is a neurodegenerative illness characterized by memory loss and other cognitive deficits. The prevalence of Alzheimer's Disease increases with age, and the presence of the disease is difficult to determine without brain biopsy. Alzheimer's Disease is characterized by the presence of neuritic plaques, neurofibrillary tangles, and neuronal cell death. Post-mortem slices of brain tissue from Alzheimer's disease patients exhibit the presence of amyloid in the form of proteinaceous extracellular cores of the neuritic plaques. The amyloid cores of these neuritic plaques are composed of a protein called the amyloid-beta or amyloid-b. Amyloid-containing neuritic plaques are a prominent feature of selective areas of the brain in Alzheimer's Disease patients as well as patients afflicted with Downs Syndrome. However, little is known about the development of Alzheimer's Disease or the mechanisms, which contribute to the disease.
  • the invention features a non-transgenic animal model for Alzheimer's Disease and methods for inducing prolonged in vivo gene expression in the brain.
  • Prolonged expression of an exogenous nucleic acid in a mammal is achieved by contacting tissue with a composition, which contains a nucleic acid, a histone, and an amphipathic compound.
  • the composition also contains a liposome.
  • the tissue is neuronal tissue, specifically central nervous system (CNS) tissue.
  • the tissue contains a post-mitotic neuronal cell, a cortical neuronal cell, a cerebellar neuronal cell, a glial cell, a vascular endothelial cell, or a hippocampal neuronal cell.
  • Gene expression in the tissue is detected in vivo for at least 48 hours after contacting the tissue with the nucleic acid composition.
  • gene expression persists for at least 72 hours, more preferably for at least 96 hours, more preferably for at least one week, and even more preferably for at least two to four weeks after contacting the tissue with the composition.
  • expression is meant transcription of a nucleic acid molecule.
  • Gene expression is measured by detecting nucleic acid transcripts, or by detecting a translation product.
  • the gene delivery system described herein has been successfully used to achieve gene expression in a target tissue for at least two months (and longer) after contacting the tissue with the composition.
  • the nucleic acid composition can be in the form of a liposome, e.g., a neutral, anionic, or cationic liposome.
  • the composition contains histone proteins, e. g., H1, H2A, H2B, H3, or H4.
  • the amphipathic compound is preferably cationic.
  • the compound is a polyamine, such as, a non-natural polyamine having a hydrophobic moiety such as a C6 C24 alkane, C6-C24 alkene, sterol, steroid, lipid, fatty acid, or a hydrophobic hormone.
  • the composition contains a nuclear localizing signal.
  • the nucleic acid encodes such as Alzheimer's Disease-associated neural thread protein 7c (AD7c-NTP)
  • the invention is useful to produce a non-human animal model of a human disease state.
  • the nucleic acid portion of the composition contains a sequence encoding an AD7c-NTP polypeptide for development of an animal model of Alzheimer's Disease.
  • the invention is useful to make a non-transgenic model for Alzheimer' Disease.
  • the model is a non-human animal, which contains an exogenous AD7c-NTP nucleic acid in its brain tissue, e. g., in a neuronal cell, of the animal.
  • AD7c-NTP gene product is over-expressed in brain tissues of the animal.
  • the animal model of human Alzheimer's Disease recapitulates the neurodegenerative process and neuropathological changes which occur in the brains of human patients with Alzheimer's Disease. Utilizing the prolonged gene expression system described above, the animal expresses an exogenous AD7c-NTP polypeptide in a neuronal cell of the animal for at least 48 hours and up to and exceeding a period of 4 weeks.
  • Cortical and/or hippocampal neuronal cells express the AD7c-NTP polypeptide over extended periods of time and exhibit physical conditions and symptoms, which mimic human Alzheimer's Disease, e. g., sporadic Alzheimer's Disease.
  • Such conditions include neuritic plaque formation, neuronal cell death by apoptosis (as well as activation of pro-apoptosis genes), and increased levels of phosphotau, APP and amyloid-b.
  • a heterologous nucleic acid or polypeptide is one that is present in an organism or location, which differs from the organism or location from which it was originally isolated.
  • a heterologous polypeptide is one encoded by a nucleic acid that has been introduced to a cell using the gene delivery methods described herein.
  • the gene therapy approach described herein leads to long term expression of therapeutic nucleic acids in adult brain tissue.
  • many approaches to gene therapy were unsuccessful because the nucleic acid formulations failed to deliver the gene to the target tissue, failed to achieve high enough levels of expression, or failed to achieve expression for long enough periods of time.
  • the nucleic acid compositions which contain a nucleic acid, a histone and an amphipathic compound, effectively deliver nucleic acids to neuronal tissue and lead to expression of the gene product for long periods of time.
  • the data described in the examples below were generated in an Alzheimer's Disease model system.
  • the data indicate that any desired gene sequence (e. g., polypeptide-encoding sequence or antisense sequences) is expressed in brain tissue for extended periods of time, e. g., even up to several months post-delivery.
  • the compositions are delivered directly into brain tissue or indirectly, e.g., intravenously.
  • the amphipathic nature of the compositions allow traversal of the blood brain barrier and access to neural tissue.
  • therapeutic polypeptides e.g., morphogens, growth factors such as nerve growth factor (NGF) or platelet-derived growth factor (PDGF), and angiogenesis inhibitors, are administered to adult neural tissue to achieve long term clinical benefit.
  • NGF nerve growth factor
  • PDGF platelet-derived growth factor
  • angiogenesis inhibitors are administered to adult neural tissue to achieve long term clinical benefit.
  • prolonged gene expression as demonstrated herein allows treatment of adult neurological disorders such as age-related neurodegeneration, Parkinson's Disease,
  • AD7c-NTP is expressed at abnormally high levels in brains with Alzheimer's Disease (compared to normal brain tissue) beginning early in the course of neurodegeneration.
  • the protein accumulates in cortical neurons and co-localizes with phospho-tau-immunoreactive cytoskeletal lesions that correlate with dementia.
  • AD7c-NTP cDNA encodes a ⁇ 41 kD membrane-spanning protein that has a hydrophobic leader sequence, a myristoylation site, and potential cleavage site. The protein is expressed on the cell surface or it may be secreted. Increased levels of AD7c-NTP protein are detectable in both cerebrospinal fluid and urine of patients with early or intermediate stages of AD.
  • Over-expression of the AD7c-NTP gene in neuronal cells in vitro produces a dimorphic phenotype characterized by either increased cell death, or neuritic sprouting in the remaining viable cells.
  • AD7c-NTP-induced neuronal cell death is mediated by apoptosis and impaired mitochondrial function and is associated with increased cellular levels of p53 and phospho-tau in AD.
  • Prolonged expression of an AD7c-NTP or NOS-3 polypeptide in brain tissue of an animal leads to a physiological state which resembles human Alzheimer's Disease.
  • Prolonged expression is achieved by administering a nucleic acid encoding AD7c-NTP or NOS-3 in a liposomal mixture with a histone protein and/or an amphipathic compound.
  • similar mixtures have been used to induce expression of nucleic acids in skeletal muscle tissue, the prolonged expression described herein has not been achieved in neuronal tissue. Long term expression of exogenous nucleic acids in neuronal tissue was surprising. The model has several advantages over transgenic models of the disease.
  • the nucleic acids are expressed in the brain and not in other tissues, and the expression can be regulated.
  • the target nucleic acid e.g., AD7c-NTP or NOS-3, is cloned under the regulation of an inducible promoter or a constitutive promoter, which preferentially directs expression of the gene product in brain tissue.
  • the model is clinically relevant in that a number of clinical indices of Alzheimer's Disease are reflected in an animal, which has been manipulated to express or over-express AD7c-NTP or NOS-3. For example, like Alzheimer's Disease brains, increased expression of phospho-tau, APP, and amyloid-b as well as plaque formation, was evident in the brains of model animals.
  • Small molecules, polypeptides, antibodies, or antibody fragments which bind to the insulin/IGF-1 domain of AD7c-NTP are used to block or inhibit signal tranduction via the IRS pathway, which in turn, inhibits neuronal cell death.
  • the inhibitory molecules block or reduce signal transduction by inhibiting binding of an endogenous ligand to the insulin/IGF-1 domain of AD7c-NTP.
  • Antibodies, which bind to AD7c-NTP have been generated using standard methods. At least 13 different hybridomas, which produce AD7c-NTP-specific antibodies, were identified. To determine whether such antibodies bind to the insulin/IGF-1 domain of AD7c-NTP, a standard binding assay is carried out using an AD7c-NTP peptide containing residues 2-14 of SEQ ID NO:2. For example, a peptide of at least 15 residues containing the insulin/IGF-1 domain of AD7c-NTP is immobilized on a solid matrix. Detectably-labelled antibody (or fragments) are allowed to bind. The antibody or antibody fragments are directly or indirectly labelled, e.g., using a radioactive, fluorescent, or colorimetric label. Unbound antibody is washed away. Retention of the labeled antibody on the solid matrix indicates that the antibody binds to the target domain.
  • a standard competitive binding assay is carried out. For example, a peptide containing the domain is immobilized. The immobilized peptide is incubated with labeled insulin or IGF-1 in the presence and absence of an antibody. A decrease in binding of insulin or IGF-1 to the immobilized peptide in the presence of the antibody (compared to the level of binding in its absence) indicates that the antibody inhibits binding of insulin or IGF-1 to the insulin/IGF-1 domain of AD7c-NTP. Blocking antibodies inhibit signal transduction, and thus, AD7c-NTP-induced neuronal death.
  • Antibodies, which bind to the insulin/IGF-1 domain of AD7c-NTP are obtained using techniques well known in the art. For example, an antibody is raised by immunizing animals with a polypeptide containing the amino acid sequence of residues 2-14 of SEQ ID NO : 2. Antibodies which bind to the domain are generated using methods known in the art, e. g., those described in U. S. Patent No. 5,863,898 . Such antibodies bind to the domain and inhibit insulin or IGF-1 binding, and therefore, signal transduction via the IRS pathway.
  • DNA encoding the antibody is cloned.
  • the cloned DNA may be used to express antibody fragments.
  • the antibody preferably binds to a site in the insulin/IGF-1 domain of AD7c-NTP.
  • the antibody binds to an epitope that is exposed on the surface of the cell.
  • the antibody is a polyclonal antisera or monoclonal antibody.
  • the disclosure encompasses not only an intact monoclonal antibody, but also an immunologically-active antibody fragment, e.g., a Fab or (Fab) 2 fragment; an engineered single chain Fv molecule; or a chimeric molecule, e. g., an antibody which contains the binding specificity of one antibody, e.g., of murine origin, and the remaining portions of another antibody, e. g., of human origin
  • antibodies e. g., AD7c-NTP monoclonal antibodies
  • AD7c-NTP monoclonal antibodies are humanized by methods known in the art.
  • Antibodies with a desired binding specificity can be commercially humanized (Scotgene, Scotland; Oxford Molecular, Palo Alto, CA).
  • compositions described herein are administered to patients diagnosed with Alzheimer's Disease as well as those who are at risk of developing Alzheimer's Disease, e. g., a person with a family history of the disease or who has been identified as having a risk factor for the disease.
  • the compositions are administered prophylactically to an elderly population (e. g., those over the age of 65,70, or 75 years of age) without evidence of clinical Alzheimer's Disease.
  • the composition is employed to ameliorate or prevent a decline in brain function associated with amyloid formation, that is less severe than dementia.
  • Prophylactic therapy is applied to persons of any age who, while displaying normal brain function, are identified as being at risk for developing Alzheimer's Disease.
  • the blood-brain barrier may be compromised in patients with neurological disease, e. g., Alzheimer's Disease, allowing systemically administered drugs to pass through the barrier into the CNS.
  • Liposome formulations of therapeutic compounds may also facilitate passage across the blood-brain barrier.
  • the formulations described herein may be administered systemically. For example, administration is accomplished intravenously, intraarterially, or intrathecally (via the spinal fluid).
  • compositions are administered locally, e.g., intraventricularly.
  • Devices for administration of compositions to an internal part of the brain are known in the art, e.g., U.S. Patent No. 5,800,390 .
  • sustained release, solid preparations and semi-solid preparations are administered directly to brain tissue.
  • Administration is carried out by inserting the needle-like member of an intracerebral device which is optionally implanted in the head so that a distal end of the guide is positioned at a site of administration.
  • compositions are administered using methods known in the art.
  • a nucleic acid encoding a therapeutic polypeptide e.g., NGF or PDGF
  • a nucleic acid encoding an antisense compound e.g., an AD7c-NTP or NOS-3 antisense template
  • a promoter which preferentially directs expression of the sequence to which it is linked in brain tissue.
  • the nucleic acid of interest is linked to a myelin basic protein (MBP) promoter, an APP promoter, a glutamine synthetase promoter, or a tyrosine hydroxylase promoter for enhanced expression of the target nucleic acid sequence in brain tissue.
  • MBP myelin basic protein
  • APP APP promoter
  • glutamine synthetase promoter glutamine synthetase promoter
  • tyrosine hydroxylase promoter for enhanced expression of the target nucleic acid sequence in brain tissue.
  • Antisense therapy is carried out by administering to an animal, e.g., a human patient, antisense template which is transcribed into an antisense RNA.
  • the antisense template is transcribed into a AD7c-NTP or NOS-3 antisense RNA.
  • the antisense RNA binds to endogenous AD7c-NTP or NOS-3 transcripts and inhibits translation of the RNA into an AD7c-NTP gene product.
  • the antisense RNA may be a short (generally at least 10, preferably at least 14 nucleotides, and up to 100 or more nucleotides) nucleotide sequence formulated to be complementary to all or a portion of a specific mRNA sequence.
  • the antisense template is preferably located downstream from the promoter sequences of the gene.
  • a poly A tail is typically located at the end of the antisense sequence to signal the end of the sequence.
  • Standard methods relating to antisense technology have been described ( Melani et al., Cancer Res. 51:2897-2901, 1991 ).
  • the antisense RNA binds to its target mRNA molecules within a cell, thereby inhibiting translation of the mRNA and down-regulating expression of the protein encoded by the mRNA.
  • the claimed DNA is introduced into target cells of an animal, e. g., a patient, using standard vectors.
  • the gene delivery systems include a histone and an amphipathic compound.
  • Suitable gene delivery systems may include liposomes, receptor-mediated delivery systems, naked DNA, and viral vectors such as herpes viruses, retroviruses, adenoviruses, and adenoassociated viruses. Methods for transfecting cells with isolated DNA are well known to those skilled in the art of molecular biology.
  • DNA are administered in a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are biologically compatible vehicles which are suitable for administration to an animal, e. g., physiological saline.
  • a therapeutically effective amount is an amount of DNA which is capable of producing a medically desirable result in a treated animal.
  • dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Dosages will vary, but a preferred dosage for intravenous administration of DNA is from approximately 10 6 to 10 22 copies of the DNA molecule.
  • the therapeutic compositions of the disclosure are administered locally or systemically. Administration will generally be intraventricularly or intravenously. The preferred form of the composition to be administered depends on the intended mode of administration and therapeutic application. For example, DNA may be administered in solution form through a catheter port directly into brain tissue.
  • Ribozyme therapy is also be used to inhibit AD7c-NTP or NOS-3 gene expression in cancer patients. Ribozymes bind to specific mRNA and then cut it at a predetermined cleavage point, thereby destroying the transcript. These RNA molecules are used to inhibit expression of the target gene according to methods known in the art ( Sullivan et al., 1994, J. Invest. Derm. 103: 85S-89S ; Czubayko et al., 1994, J. Biol. Chem. 269: 21358-21363 ; Mahieu et al, 1994, Blood 84: 3758-65 ; Kobayashi et al. 1994, Cancer Res. 54: 1271-1275 ).
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site are evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable. The suitability of candidate targets are evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays. Antisense RNA and DNA molecules and ribozymes are prepared by methods known in the art, such as chemically synthesis of oligodeoxyribonucleotides using solid phase phosphoramidite techniques.
  • Assays to identify a compound which inhibits Alzheimer's Disease-associated neuronal cell death are carried out by culturing an AD7c-NTP over-expressing cell with a candidate compound and measuring cell viability. Cell viability is measured using methods known in the art, e.g., vital dye exclusion or incorporation of tritiated thymidine. An increase in cell viability in the presence of the compound compared to in its absence indicates that the compound inhibits Alzheimer's Disease associated neuronal cell death.
  • the cell is a primary or immortalized cell line.
  • Primary cerebellar neuronal cell, hippocampal cell, glial cell, or vascular endothelial cells which over-express AD7c-NTP encoding DNA or NOS-3 encoding DNA in an inducible or constitutive manner are used in the assay.
  • the cells are in the form of neuronal tissue explants from an Alzheimer's Disease patient or Alzheimer's Disease model animal.
  • a non-human animal model for the disease such as the model described herein in which AD7c-NTP is overexpressed, is contacted with a candidate compound and neuronal cell viability measured. An increase in cell viability in the presence of the compound compared to in its absence indicates that the compound inhibits Alzheimer's Disease associated neuronal cell death.
  • Candidate compounds such as organic compounds, polypeptides, or antibodies and fragments thereof, are administered systemically or directly to brain tissue of the animal.
  • APP expression is detected in the tissue using known methods such as immunohistochemical analysis. A decrease in APP expression in the presence of the compound compared to in its absence indicates that the compound inhibits a symptom or condition of Alzheimer's Disease.
  • the animal model of Alzheimer's Disease described herein develops senile neuritic plaques similar to those detected in autopsy samples of human brains from Alzheimer's Disease patients.
  • Compounds which inhibit the formation or progression of plaques are identified by contacting the non-human animal model with a candidate compound and detecting amyloid plaques in the tissue. Plaques are detected using known methods such as histological and immunohistological evaluation of brain tissue and vascular tissue in the brain. A decrease in the amount of plaques in the presence of the compound compared to in its absence indicates that the compound inhibits senile plaque formation.
  • Compounds which bind to the insulin/IGF-1 domain of AD7c-NTP block binding of insulin or IGF-1 to AD7c-NTP and inhibit signal transduction.
  • Such compounds are identified using standard methods. For example, candidate polypeptides, antibodies, or other compounds are immobilized, e.g, on a chromatographic column or on another solid matrix such as a microarray or microtiter plate. The immobilized compounds are contacted with detectably-labelled polypeptide (e.g., a polypeptide containing residues 2-14 of SEQ ID NO:2). Unbound peptide is washed away, and the label is detected.
  • Compositions which bind to the insulin/IGF-1 domain of AD7c-NTP are identified by retention of the detectably-labeled AD7c-NTP polypeptide.
  • a competitive binding assay is carried out.
  • a polypeptide containing residues 2-14 of SEQ ID NO:2 is immobilized and contacted with insulin or IGF-1 in the presence and absence of a candidate compound.
  • the candidate compound is an AD7c-NTP antibody or an antibody fragment of an AD7c-NTP-specific antibody.
  • the insulin or IGF-1 may be detectably labelled.
  • a decrease in binding of insulin or IGF-1 to the immobilized AD7c-NTP peptide (as detected by presence of label) in the presence of the candidate compound compared to the level of binding in the absence of the compound indicates that the candidate compound inhibits binding of insulin or IGF-1 to the insulin/IGF-1 hybrid domain of AD7c-NTP.
  • Such inhibitory compounds inhibit signal transduction via the insulin/IGF-1 signal transduction pathway, and thus, inhibit a symptom or condition of Alzheimer's Disease.
  • AD7c-NTP-induced neuronal cell death associated with Alzheimer's Disease is mediated by apoptosis. Apoptotic death is associated with increased cellular levels of p53 and phospho-tau, as occur in AD. Accordingly, another method of screening for therapeutic agents is carried out by contacting AD7c-NTP over-expressing cells (or NOS-3 over-expressing cells) with a candidate compound in vitro or in vivo and measuring the level of p53 and phospho-tau in the cells. A decrease in the level of p53 or phospho-tau in the presence of the candidate compound compared to the level in the absence of the candidate compound indicates that the candidate compound reduces neuronal cell death associated with Alzheimer's Disease.
  • Example 1 AD7c-NTP Induced Apoptosis and Sprouting
  • AD7c-NTP neuronal thread protein gene is over-expressed in Alzheimer's disease beginning early in the course of disease.
  • AD7c-NTP protein accumulates in cortical neurons and co-localizes with phospho-tau-immunoreactive cytoskeletal lesions.
  • Over-expression of the AD7c-NTP gene results in a dimorphic phenotype associated with both apoptosis and enhanced neuritic sprouting.
  • An inducible mammalian expression vector was used to regulate AD7c-NTP expression in PNET2 neuronal cells, and the effects of insulin (50 nM), IGF-1 (5 ng/ml), NGF (2.5 ng/ml) or PDGF (5 ng/ml) stimulation on cellular morphology, gene expression, and intracellular signaling pertinent to AD-type neurodegeneration was examined. Cells transfected with the CAT gene were used as negative controls.
  • Insulin or IGF-1 stimulation of cells induced to express AD7c-NTP resulted in increased cell death, increased levels of p53, p21/Waf1, phospho-JNK, nitric oxide synthase-3, phospho-tau, and the p25 regulatory partner of Cdk 5, and inhibition of Bcl2 expression.
  • cells stimulated with NGF or PDGF despite high levels of AD7c-NTP expression, exhibited prominent neuritic sprouting, and high levels of Bcl-2 and phospho-Erk MAPK.
  • Clones were transfected with a second vector (pOPRSV1) carrying the AD7c-NTP cDNA or the chloramphenicol acetyltransferase (CAT) gene, and stable clones were selected with hygromycin and G418.
  • the second vector contains an RSV promoter that drives expression of the gene of interest, and an ideal operator sequences for Lac repressor binding.
  • Isopropyl-1- ⁇ -D-thiogalactopyranoside (IPTG) stimulation (1-5 mM) turns off the Lac repressor protein and induces expression of CAT or AD7c-NTP within 4-8 hours. After induction, gene expression persists for 48-96 hours, and is rapidly inhibited by withdrawal of IPTG. Exploratory studies demonstrated that 3 mM IPTG was optimum for inducing gene expression in the clones.
  • the cells were serum-starved for 16 hours, after which IPTG (1-5 mM) was added to the medium. Four hours later, the cells were stimulated with insulin (50 nM), IGF-1 (5 ng/ml), NGF (5 ng/ml), or platelet-derived growth factor (PDGF; 5 ng/ml) for 24-72 hours. The cells were analyzed for morphological changes, viability, AD7c-NTP immunoreactivity, and expression of pro-apoptosis genes, survival genes, and phospho-tau.
  • insulin 50 nM
  • IGF-1 5 ng/ml
  • NGF 5 ng/ml
  • PDGF platelet-derived growth factor
  • Viability was measured by a standard crystal violet assay. Crystal violet dye labels only viable cells. The assays were performed with cells seeded into 96-well plates at a density of 2 x 10 4 cells/well. The absorbances were measured using a Spectracount plate reader (Packard, Meriden, CT). The crystal violet absorbances increased linearly with cell density between 10 4 and 5x10 5 cells/well.
  • Standard Western blot assays wew used to measure cellular levels of p53, Bcl-2, p21/Waf1, phospho-tau, tau, c-fos, NTP, and activated (phosphorylated) forms of Erk mitogen activated protein (MAP) kinase, amino-terminal c-jun-activated kinase (pJNK), and p38/HOG1.
  • MAP Erk mitogen activated protein
  • pJNK amino-terminal c-jun-activated kinase
  • p38/HOG p38/HOG1.
  • Samples containing 60 ⁇ g of protein were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to PVDF membranes, and analyzed by Western immunoblotting. Immunoreactivity was detected with horseradish peroxidase conjugated secondary IgG and PicoWest enhanced chemiluminescence reagents (Pierce Chemical Company, Rockford, IL). CAT activity was measured using known methods.
  • the MICE assay is known in the art (e.g., as described by de la Monte et al., 1999, Biotechniques 26:107301076 ).
  • the assay is a rapid and sensitive method for quantifying immunoreactivity in 96-well micro-cultures and combines the advantages of the enzyme-linked immunosorbant assay with immunocytochemical staining to permit sensitive in situ quantification of protein expression with values normalized to cell density.
  • the cells were fixed in Histochoice (Amresco, Solon, Ohio), permeabilized with 0.05% saponin in Tris-buffered saline (50 mM Tris, pH 7.5, 0.9% NaCl; TBS), and blocked with Superblock-TBS (Pierce, Rockford, IL).
  • the cells were then incubated overnight at 4°C with primary antibody diluted in TBST-BSA. Immunoreactivity was detected using horseradish peroxidase conjugated secondary antibody (Pierce, Rockford, IL) and the TMB soluble peroxidase substrate (Pierce, Rockford, IL). Absorbances were measured at 450 nm using a Spectracount plate reader.
  • the cells were harvested in Triton lysis buffer supplemented with protease and phosphatase inhibitors.
  • Total PI3K was immunoprecipitated from 500 ⁇ g protein in cell lysates using rabbit polyclonal antibodies to the p85 subunit of PI3K and Protein A sepharose.
  • the immunoprecipitants were incubated for 5 minutes at room temperature with 10 ⁇ g of sonicated phosphatidylinositol in HEPES buffer (200 mM HEPES, 4 mM EGTA, 4 mM sodium phosphate, pH 7.0).
  • Monoclonal antibodies to p53 and p21/Waf1, and polyclonal antibodies to c-fos were obtained from Oncogene Research Products (Cambridge, MA).
  • Rabbit polyclonal antibody to the insulin receptor substrate -1 and the p85 subunit of PI3K was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY).
  • Polyclonal antibody to Bcl-2 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and antibodies to both total and activated JNK, Erk MAPK, and p38/HOG1 were obtained from Promega Corp. (Madison, WI).
  • Anti-tau was purchased from Dako Corp. (Carpenteria, CA).
  • Protein A sepharose was purchased from Amersham-Pharmacia Biotechnology (Arlington Heights, IL). Recombinant human NGF, PDGF, and IGF1 were purchased from Sigma Co (St. Louis, MO). Human insulin was purchased from Novagen.
  • Data described in this example represent the means ⁇ S.D.'s generated with results obtained from 3 to 6 experiments. Inter-group comparisons were made using Student t-tests or analysis of variance (ANOVA) with Fisher least significant difference (LSD) post-hoc tests.
  • PNET2 cells were stably transfected with the AD7c-NTP cDNA using the LacSwitch II vector system (Stratagene, La Jolla, CA) in which gene expression was regulated by the LacZ promoter and induced by IPTG stimulation (1-5 mM).
  • Control cells were similarly transfected with a cDNA that encodes the chloramphenicol acetyl transferase (CAT) gene.
  • CAT chloramphenicol acetyl transferase
  • the clones selected for study exhibited tight regulation of gene expression since in the absence of IPTG, AD7c-NTP expression or Cat activity were either very low level or undetectable. IPTG stimulation induced AD7c-NTP gene expression or CAT activity, which was sustained for up to 96 hours.
  • control cells did not exhibit IPTG-inducible NTP expression, and GAPDH expression was also unchanged by IPTG stimulation.
  • CAT activity was substantially increased by IPTG induction of the CAT reporter gene in those cells.
  • AD7c-NTP gene resulted in reduced neuronal cell viability and neuritic sprouting in the same cultures.
  • Analysis of the amino acid subsequence of the translated AD7c-NTP cDNA indicated the presence of an insulin/IGF1 hybrid domain (amino acids 2-14 of SEQ ID NO:2), which suggested that the effects of AD7c-NTP over-expression might be linked to growth factor stimulation. Therefore, studies were undertaken to determine if the morphological features of neuronal cells that over-express the AD7c-NTP cDNA were regulated by differential responses to growth factors.
  • IPTG IPTG
  • IGF-1 IGF-1
  • NGF 2.5 ng/ml
  • PDGF PDGF
  • PNET2 cells could be maintained in serum-free medium for 5 to 7 days with insulin, IGF-1, NGF, or PDGF as the only growth stimulus.
  • each of these growth factors supports DNA synthesis and promotes modest outgrowth of multipolar neurites in PNET2 cells.
  • the CAT-expressing control cells exhibited modest degrees of neuritic sprouting with insulin, IGF-1, PDGF, or NGF stimulation in non-transfected PNET2 cells.
  • PNET2 cells that were induced to express AD7c-NTP manifested different morphological responses depending upon the growth factor used. Insulin stimulation resulted in neurite retraction, cell rounding and increased cell death (floating and refractile), whereas PDGF or NGF stimulation resulted in extensive neuritic growth with multipolar fine interconnecting processes and minimal evidence of on-going cell death.
  • NGF stimulation had the same effect on cellular morphology as PDGF.
  • the IGF-1 stimulated cultures contained two populations of cells: some were rounded, refractile, and floating, while others were cytologically intact and exhibited prominent neuritic processes following FCS stimulation of AD7c-NTP transfected cells. It is noteworthy that IGF-1 is a major growth factor present in FCS.
  • Apoptosis of PNET2 cells was found to be mediated by increased levels of p53 and p21, reduced levels of Bcl-2.
  • the levels of p53, Bcl-2, and p21/Waf-1 expression was measured by Western blot analysis or the MICE assay.
  • the effects of PDGF and NGF were similar. Stimulation with insulin, IGF-1 or PDGF resulted in similar levels of the ⁇ 41 kD AD7c-NTP protein as demonstrated by Western blot analysis.
  • AD7c-NTP cyclin dependent protein kinase 5
  • IPTG induction of the CAT gene did not result in increased expression of any of these molecules, regardless of which growth factor was used to stimulate the cells.
  • Western blot analysis demonstrated that cells which were induced to express AD7c-NTP and stimulated with insulin or IGF-1 exhibited increased levels of phospho-tau ( Figs, 3A-C ), NOS-3, and p25 relative to corresponding cultures stimulated with PDGF, whereas the levels of tau and Cdk5 protein were not modulated in relation to growth factor stimulation.
  • the membranes were probed using antibodies to Erk, phospho-Erk, Jun, and phospho-JNK. While the levels of total Erk and Jun were found to be similar for all samples, striking differences in the levels of growth factor stimulate phospho-Erk and phospho-JNK were detected. Insulin and IGF-1 stimulation resulted in increased levels of phosph-Erk p42/p44, with peak levels detected within10 or 15 minutes after adding these growth factors to the medium, whereas PDGF had minimal effect on the levels of phospho-Erk MAPK in short-term stimulation experiments.
  • PI3 kinase is an important mediator of neuronal survival.
  • the pro-survival effects of PI3K are mediated through activation of Akt (protein kinase B) and phosphorylation of Bad to render it inactivated.
  • Inhibition of PI3 kinase activity is associated with PNET2 cell apoptosis. Therefore, experiments were carried out to determine if growth factor stimulated PI3 kinase activity was inhibited in insulin or IGF-1-stimulated cultures. Such inhibition has been observed following chronic ethanol exposure.
  • IRS-1 insulin-receptor substrate-1
  • IRS-1-independent pathways both IRS1-associated PI3 kinase and total PI3 kinase activities were measured.
  • IRS-1-associated PI3 kinase activity was sharply increased by insulin but not PDGF stimulation. High levels of IRS-1-associated PI3 kinase activity were detected throughout the 30 minutes of insulin stimulation, whereas in the PDGF-stimulated cultures, IRS-1-associated PI3 kinase activity was virtually undetectable.
  • PI3 kinase activity was examined by immunoprecipitation/Western blot analysis.
  • Levels of PI3 kinase activity were measured in anti-phospho-tyrosine (PY) immunoprecipitates.
  • PY anti-phospho-tyrosine
  • the immunoprecipitation/Western blotting studies demonstrated similarly high levels of PY-associated p85 (subunit of PI3K), both after 5-30 minutes and 24 or 48 hours of growth factor stimulation.
  • PI3 kinase activity was readily detected in the PY immunoprecipitates prepared with cells stimulated with insulin or PDGF.
  • AD7c-NTP gene is over-expressed in brains with Alzheimer's disease at early and intermediate stages of neurodegeneration.
  • AD7c-NTP immunoreactivity co-localizes with early phospho-tau immunoreactive cytoskeletal lesions in brains with AD, and increased levels of phospho-tau and AD7c-NTP in cerebrospinal fluid correlate with the severity of Alzheimer's Disease dementia.
  • AD7c-NTP Prior to the invention, it was difficult to investigate the role of AD7c-NTP using standard stably transfected clones due to progressive depletion of the cells in culture.
  • AD7c-NTP inducible mammalian expression vector system
  • human CNS-derived PNET2 neuronal cells in which gene expression was induced by the addition of IPTG to the medium, and inhibited by withdrawal of IPTG.
  • IPTG-induced gene expression AD7c-NTP or CAT activity
  • AD7c-NTP or CAT activity IPTG-induced gene expression was detectable within 8 hours and sustained for up to 96 hours.
  • the effects of AD7c-NTP over-expression was examined using 6 clones that had tightly regulated inducible gene expression whereby in the absence of IPTG, expression of the ⁇ 41 kD N3I4-immunoreactive AD7c-NTP protein was virtually undetectable.
  • IPTG stimulation substantially increased AD7c-NTP expression as demonstrated by Western blot analysis and the MICE assay.
  • the expression of a non-relevant gene, GAPDH was unchanged by IPTG stimulation.
  • the expected ⁇ 41 kD AD7c-NTP protein was easily detected by Western blot analysis, whereas in control cells induced to express the CAT gene, AD7c-NTP immunoreactivity was either very low level or undetectable.
  • the MICE assay was used to quantify AD7c-NTP expression in the presence or absence of IPTG stimulation.
  • the MICE assay provides a more sensitive measure of immunoreactivity and permits simultaneous analysis of multiple cultures under the same conditions without requiring protein extraction or gel electrophoresis. Importantly, the levels of immunoreactivity are corrected for cell density in the calculation of the MICE index.
  • MICE assay we confirmed the presence of very low levels of N3I4-immunoreactive AD7c-NTP in un-induced PNET2 cells, and a greater than five-fold increase in the mean levels of N3I4-immunoreactive AD7c-NTP expression after 24 hours of IPTG induction of gene expression.
  • the increased PNET2 cell death observed with insulin or IGF1 stimulation was associated with increased expression of the p53 pro-apoptosis gene product and reduced expression of the Bcl-2 survival gene.
  • higher levels of p21/Waf1 through which p53 frequently signals to mediate apoptosis, occurred in insulin and IGF-1 stimulated cultures.
  • the insulin-stimulated cells also had increased levels of phospho-JNK relative to IGF-1- and PDGF-stimulated cultures, both in the short-term (5-60 min) and long-term (24 hrs) stimulation studies.
  • p53-induced apoptosis can be mediated by signaling through the jun B/JNK pathway.
  • AD7c-NTP over-expression and insulin or IGF1 stimulation mimic responses associated with oxidative stress.
  • PNET2 neuronal cell death was also associated with increased levels of nitric oxide synthase-3 (NOS3), phospho-tau, and the p25 regulatory partner of cyclin dependent kinase 5 (Cdk-5).
  • NOS3 expression like AD7c-NTP, is aberrantly increased in Alzheimer's Disease cortical neurons and dystrophic neurites beginning early in the course of neurodegeneration.
  • Intra-neuronal phospho-tau-immunoreactive cytoskeletal lesions are a major hallmark of Alzheimer's Disease-associated neurodegeneration as their densities correlate with severity of dementia.
  • Cdk5 activity is increased due to increased neuronal levels of p25.
  • Cdk5 kinase activity is regulated by interaction with p35, or its truncated, catalytically active C-terminal fragment, p25.
  • p35 has a short half-life, which may be important for the on-off regulation of Cdk5 kinase activity.
  • p25 is highly stable and over-expression of p25 leads to constitutive activation of Cdk5 kinase and also promotes apoptosis.
  • Tau and neurofilament proteins can be phosphorylated by Cdk5 kinase, and in brains with Alzheimer's Disease, p25 immunoreactivity and cdk5 kinase activity co-localize with degenerating neurons and cell processes that contain phospho-tau immunoreactive cytoskeletal lesions. Therefore, over-expression of AD7c-NTP in neuronal cells stimulated with insulin or IGF-1 results in increased levels of both phospho-tau and p25, which have already been mechanistically linked to AD-type neurodegeneration.
  • NOS-3 over-expression is a feature of Alzheimer's Disease as well as other neurodegenerative diseases. High levels of NOS-3 can lead to increased levels of NO and oxidative injury, and some data indicate that oxidative injury to neuronal cells can promote tau phosphorylation and the conversion of p35 to p25. Constitutive over-expression of the NOS-3 cDNA in PNET2 neuronal cells is sufficient to cause apoptosis associated with increased levels of p53, reduced levels of Bcl-2, increased p35-to-p25 conversion, and tau phosphorylation.
  • AD7c-NTP-induced neuronal apoptosis is mediated by oxidative injury due to attendant increases in the levels of NOS-3 expression.
  • NOS-3 and AD7c-NTP Aberrant expression of both NOS-3 and AD7c-NTP begins early, involves the same structures, and is intimately associated with apoptotic cell loss and neurodegeneration.
  • the AD7c-NTP protein has a hydrophobic leader sequence (residues 1-15 of SEQ ID NO:2) and at least one transmembrane domain. For example, seven putative transmembrane domains are located within the AD7c-NTP protein (residues 70-92, 111-18, 126-134, 147-177, 182-191, 241-249, and 339-351 of SEQ ID NO:2).
  • the insulin/IGF1 chimeric receptor motif is located at the amino terminus (residues 2-14 of SEQ ID NO:2) and can be exposed to the cell surface, rendering it accessible to interactions with extracellular growth factors.
  • insulin and IGF-1 signaling through one of the insulin receptor substrates (IRS) such as IRS-1 or IRS-2 which are both expressed in neuronal cells may mediate the observed effects in the context of AD7c-NTP over-expression.
  • IRS insulin receptor substrates
  • insulin stimulation of ethanol exposed neuronal cells causes apoptosis
  • NGF stimulation rescues the cells from ethanol-induced apoptosis and also promotes neuritic sprouting.
  • AD7c-NTP over-expression The mechanisms involved in the insulin-stimulated pro-apoptosis signaling and NGF-mediated rescue are distinct from those identified in relation to AD7c-NTP over-expression. Since insulin, IGF-1, NGF, and PDGF can support growth and promote neuritic sprouting in PNET2 cells, the data described herein indicates that AD7c-NTP over-expression alters or impairs insulin- and IGF-1-stimulated signaling mechanisms in neuronal cells. The adverse effects of AD7c-NTP expression on intracellular signaling appear to be selective with respect to growth factor stimulated neuronal survival and anti-apoptosis mechanisms.
  • AD7c-NTP gene causes both increased cell death and neuritic sprouting, which are two of the prominent abnormalities associated with Alzheimer's Disease. These results indicate that aberrantly increased AD7c-NTP expression has a role in Alzheimer's Disease-type neurodegeneration.
  • Primary postmitotic neurons were efficiently transfected with conventional recombinant plasmid DNA to evaluate the effects of gene over-expression in the context of neurodegeneration using art-recognized in vitro models.
  • Post-mitotic primary rat cerebellar neuron (rCBN) cultures were generated with brain tissue derived from postnatal day 6 pups.
  • Five-day-old cultures were transfected with the full length AD7c-NTP cDNA (pcDNA3-AD7c) or the luciferase (pcDNA3-Luc) or LacZ (pcDNA3-beta-Gal) reporter gene ligated into the pcDNA3.1 vector (Invitrogen) in which gene expression was regulated by a CMV promoter.
  • Cells seeded into 6-well or 96-well plates were transfected using IT-100 or LT-1 Mirus transfection reagent (Panvera) following the manufacturer's protocol.
  • Transfection efficiency ranged from 10% to 25% as demonstrated by co-transfection with recombinant plasmid DNA expressing green fluorescent protein (pcDNA3-GFP) and visualizing the percentage of labeled cells by fluorescence microscopy. The cells were analyzed for gene expression, viability, and morphology 24, 48, or 72 hours after transfection.
  • pcDNA3-GFP green fluorescent protein
  • Viability was measured by a standard crystal violet assay.
  • the assays were performed with cells seeded into 96-well plates at a density of 2 x 104 cells/well.
  • the absorbances were measured using a Spectracount plate reader (Packard, Meriden, CT).
  • the crystal violet absorbances increased linearly with cell density between 104 and 5x105 cells/well.
  • the MICE assay is a rapid and sensitive method for quantifying immunoreactivity in 96-well micro-cultures and combines the advantages of the enzyme-linked immunosorbant assay with immunocytochemical staining to permit sensitive in situ quantification of protein expression with values normalized to cell density. Briefly, the cells were fixed in Histochoice (Amresco, Solon, Ohio), permeabilized with 0.05% saponin in Tris-buffered saline (50 mM Tris, pH 7.5, 0.9% NaCl; TBS), and blocked with Superblock-TBS (Pierce, Rockford, IL). The cells were then incubated overnight at 4°C with primary antibody diluted in TBST-BSA.
  • Immunoreactivity was detected using horseradish peroxidase conjugated secondary antibody (Pierce, Rockford, IL) and the TMB soluble peroxidase substrate (Pierce, Rockford, IL). Absorbances were measured at 450 nm using a Spectracount plate reader (Packard Instrument Company, Meriden, CT). Relative culture cell density was determined by subsequently staining the cells with 0.1% Coomassie blue dye, lysing the labeled cells with 1% SDS, and measuring the absorbances of at 540 nm. The MICE index was calculated from the ratio of the absorbances measured for immunoreactivity and cell density. Eight or16 replicate culture wells were analyzed in each experiment. All experiments were repeated at least 3 times.
  • Fig. 5 demonstrates luciferase activity in rCBN cultures transfected with pcDNA3-Luc. Increased luciferase activity was detected 48 hours after transfection, and at the 72-hour time point, the levels were further increased.
  • Western blot analysis using an AD7c-NTP specific monoclonal antibody generated to recombinant AD7c-NTP protein demonstrated increased levels of the ⁇ 41 kD AD7c-NTP protein in cells transfected with pcDNA3-AD7c relative to cells transfected with pcDNA3-Luc ( Fig. 6A ).
  • the data described herein demonstrates that efficient gene transfer in postmitotic neurons was achieved using a formulation containing a nucleic acid, a histone protein, liposomes, and an amphipathic compound.
  • the DNA is delivered using the MIRUS polyamine transfection reagent.
  • Over-expression of the AD7c-NTP gene causes neuronal cell death and neuritic sprouting in postmitotic neurons.
  • optimum gene expression following transfection was detected after 72 hours rather than 24 or 48 hours, as generally occurs in transfected cell lines.
  • the advantage of using the polyamine transfection composition is that studies can now be done with primary neuronal cell cultures rather than transformed cell lines.
  • AD7c-NTP transfection of post-mitotic neurons with pcDNA3-AD7c causes increased cell death and neuritic sprouting.
  • Over-expression of the AD7c-NTP gene was documented by Western blot analysis, the MICE assay, and immunocytochemical staining using monoclonal antibodies generated to the human recombinant protein.
  • transfection with non-relevant genes was associated with very low-level or undetectable AD7c-NTP expression.
  • AD7c-NTP Over-expression of AD7c-NTP resulted in neuronal cell death and prominent neuritic sprouting. These two phenotypes were originally observed in PNET2 neuronal cell lines that were transiently transfected with the same cDNA. However, PNET2 cells are immature, proliferative, and transformed. To determine the potential role of the AD7c-NTP gene in relation to AD-type neurodegeneration which affects post-mitotic neurons exclusively, it was necessary to demonstrate that over-expression of the AD7c-NTP gene has the same effect in post-mitotic neurons as observed previously in PNET2 cells.
  • phase contrast microscopy studies demonstrated progressive depletion of granule cell neurons, and prominent growth of long, thin interconnecting processes from the remaining viable neurons in the AD7c-NTP transfected cultures.
  • the neurite outgrowth was probably not just a response to cell loss since rCBN cultures treated with oxidants exhibit similar degrees of cell loss as well as neurite retraction rather than sprouting.
  • Over-expression of the AD7c-NTP gene may cause neuronal cell death, while exposure of neurons to extracellular (secreted) AD7c-NTP protein may promote neuritic sprouting.
  • AD dementia is due to cell loss, mediated by apoptosis, impaired mitochondrial function, and possibly necrosis.
  • a second major correlate of dementia is synaptic disconnection due to neuritic degeneration.
  • AD7c-NTP is over-expressed in brains with sporadic AD. Increased levels of the corresponding ⁇ 41 kD protein are detectable at early and intermediate stages of AD, and in the brain, AD7c-NTP immunoreactivity co-localizes with phospho-tau.
  • Over-expression of the AD7c-NTP gene causes apoptosis, impaired mitochondrial function, and aberrant neuritic sprouting in cultured neuronal cells.
  • AD7c-NTP In vivo gene transfer methods were used to demonstrate that over-expression of AD7c-NTP in the brain causes neuronal cell loss associated with activation of pro-apoptosis genes and increased levels of phospho-tau, amyloid precursor protein, amyloid-b, and nitric oxide synthase-3 expression localized in neurons or senile plaque-like structures.
  • Over-expression of AD7c-NTP leads to increased levels of NOS-3, which promotes chronic oxidative stress and secondary neurodegenerative changes similar to the abnormalities observed in AD.
  • DNA was purified using endotoxin-free columns (Qiagen Inc., Valencia, CA). DNA (3 ⁇ g/inoculum) was formulated with a histone and an amphipathic compound (e.g., Transit In Vivo polyamine reagent (Panvera Inc., Madison, WI)). The DNA mixture was injected into the right lateral ventricle of a rat brain in a volume of 25 ⁇ l using a stereotactic frame and atlas. The rats were sacrificed to examine gene expression and histopathology, 1, 2, 3, 4, 7, 14, 21, or 28 days after the inoculation.
  • amphipathic compound e.g., Transit In Vivo polyamine reagent (Panvera Inc., Madison, WI)
  • Western blot analysis was used to measure AD7c-NTP protein expression in cell lysates prepared in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease and phosphatase inhibitors 7 . Protein concentrations were measured using the BCA assay (Pierce Chemical Company, Rockford, IL). Samples containing 60 ⁇ g of protein were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to PVDF membranes, and analyzed by Western immunoblotting. Immunoreactivity was detected with horseradish peroxidase conjugated secondary IgG and PicoWest enhanced chemiluminescence reagents (Pierce Chemical Company, Rockford, IL), and quantified using the Kodak Digital Imager System.
  • RIPA radioimmunoprecipitation assay
  • the brains were sectioned in the coronal plane and either embedded in paraffin or snap frozen for cryosectioning. Histopathological studies were performed on paraffin sections stained with hematoxylin and eosin. Immunoreactivity for AD7c-NTP, pro-apoptosis genes (p53, Bax), phospho-tau, amyloid precursor protein (APP), amyloid- ⁇ (A ⁇ ), synaptophysin, or nitric oxide synthase-3 (NOS3) was detected in adjacent paraffin sections using known methods. ⁇ -galactosidase activity was detected in cryostat sections using standard methods.
  • Gene transfer was made by inoculating the right lateral ventricle with recombinant plasmid DNA complexed with histone proteins and an amphipathic compound.
  • the cerebral hemispheres were hemisected and analyzed separately for AD7c-NTP expression by Western blot analysis using an AD7c-NTP-specific monoclonal antibody (e.g., N3I4), which binds to the recombinant protein.
  • an AD7c-NTP-specific monoclonal antibody e.g., N3I4
  • FIG. 8A depicts expression of the ⁇ 41 kD AD7c-NTP protein in control brains (ipsilateral to injection), and brain tissue contralateral (left side) and ipsilateral (right side) to the gene transfer.
  • the graph shown in Fig. 8B illustrates the densitometric analysis results obtained using the Kodak Digital Science Image Station. The levels of immunoreactivity are expressed in arbitrary luminescence units.
  • AD7c-NTP expression Over time, the levels of AD7c-NTP expression also increased in the left cerebral hemisphere, contralateral to the side of inoculation, and although the peak levels observed after 14 days were below those measured on the right side ( Fig. 8A ). These results were reproduced in three separate experiments using at least 6 animals per group.
  • Neuronal cell loss was detectable in brains harvested up to 4 weeks after pAD7c-NTP gene transfer. Control brains harvested over the same interval consistently exhibited normal neuronal morphology similar to un-inoculated specimens. These data indicate that rodent brains inoculated with pAD7c-NTP exhibited neuronal loss with scattered pale, irregular neurons that lacked nuclei (ghost cells), as well as evidence of a cluster of dying neurons. These features mimic the features in human Alzheimer's Disease.
  • AD7c-NTP Over-expression of AD7c-NTP resulted in increased neuronal apoptosis and apoptosis proneness, manifested by higher densities of TUNEL+ nuclei, and increased immunoreactivity corresponding to the p53 and Bax pro-apoptosis gene products ( Figs. 10A-D , Figs. 11A-C , and Figs. 12A-C ).
  • TUNEL-positive labeling of neuronal nuclei was observed in the cerebral cortex following AD7c-NTP gene transfer compared to the control (rat brains inoculated with the Luciferase control gene). Tissue sections examined 1 week after gene transfer into the right lateral ventricle and cerebral hemisphere. TUNEL labeling of nuclei reflects increased genomic DNA nicking and fragmentation as occur prior to apoptosis.
  • the damaged cells were identified as neuronal based upon their pyramidal shape, large size (> 8 microns diameter), distribution within the cerebral cortex, and positive labeling with anti-Hu in adjacent sections.
  • the increased apoptosis (TUNEL+) and pro-apoptotic gene (p53, Bax) expression were detected one week after pAD7c-NTP inoculation.
  • the levels and distribution of cellular labeling were initially higher ipsilateral to the inoculations, but after two weeks, the two hemispheres were somewhat similar in these regards, corresponding with the more symmetrical distribution of AD7c-NTP expression, as demonstrated by Western blot analysis.
  • AD7c-NTP e.g., the N2U6
  • NTP neuronal thread protein
  • AD7c-NTP over-expression To determine the effects of AD7c-NTP over-expression on other genes and proteins previously linked to neurodegeneration, adjacent sections immunostained with antibodies to phospho-tau, amyloid precursor protein, amyloid-b peptide, and synaptophysin.
  • Figs. 14A-D All brains inoculated with pAD7c-NTP exhibited increased levels of phospho-tau localized in neurons and neuritic plaque-like structures.
  • Increased phospho-tau immunoreactivity was often abnormally localized in neuronal nuclei in the cerebral cortex of brains expressing exogenous AD7c-NTP.
  • the phospho-tau-immunoreactive plaques were distributed in the cerebral cortex and in deep gray matter structures. Control brains exhibited only diffuse low levels of phospho-tau immunoreactivity and no evidence of neuritic plaque formation.
  • pTau phospho-Tau
  • Tissue sections wereexamined 2 weeks after gene transfer into the right lateral ventricle and cerebral hemisphere.
  • the brains inoculated with the AD7c-NTP cDNA also exhibited pTau-immunoreactive neuritic plaques.
  • APP amyloid precursor protein
  • Fig. 15A,B cortical neurons
  • Fig. 15C hippocampal neurons, some of which were degenerating
  • Fig. 15C plaque-like structures that had the appearance of dense-cores
  • Figs. 15D, E neuritic structures
  • the plaque-like lesions were scattered among APP-positive cortical neurons, but some appeared to be associated with small vessels.
  • Control brains exhibited APP immunoreactivity in the choroid plexus and initially (24-72 hours after inoculation) adjacent to the injections site, but not 1 or 2 weeks post gene transfer ( Fig. 15E ).
  • AD7c-NTP Alzheimer's Disease associated neurodegeneration
  • NOS-3 expression in increased in cortical neurons in brains with Alzheimer's Disease beginning early in the course of neurodegeneration.
  • Abnormally increased levels of NOS-3 immunoreactivity in brains with Alzheimer's Disease as well as other forms of neurodegeneration are associated with apoptosis and apoptosis proneness mediated in part by increased production of peroxynitrite.
  • AD7c-NTP and NOS-3 expression begin at approximately the same time period in Down syndrome, which represents a natural human model of Alzheimer's Disease associated neurodegeneration, studies were undertaken to determine if NOS-3 expression was linked and downstream of AD7c-NTP.
  • AD7c-NTP Induced Neurodegeneration following in vivo gene transfer
  • AD7c-NTP results in neurodegeneration characterized by increased neuronal cell death mediated by apoptosis and impaired mitochondrial function, and associated increased expression of phospho-tau and the p53 pro-apoptosis gene.
  • long term expression of this gene or other genes has not been possible until the present demonstration thereof
  • the gene transfer system described herein provides a method for inducing prolonged in vivo gene expression in non-skeletal muscle tissue using a formulation, which contains a nucleic acid, a histone protein, a liposome, and an amphipathic compound.
  • the system was used to examine the effects of AD7c-NTP over-expression in vivo.
  • Western blot analysis revealed substantially higher levels of the expected ⁇ 41 kD AD7c-NTP protein in brains inoculated with pAD7c-NTP compared with brains inoculated with pLuc or pLacZ controls.
  • AD7c-NTP causes apoptosis by activating pro-apoptosis mechanisms as observed in brains with AD.
  • brains inoculated with pAD7c-NTP exhibited increased expression of phospho-tau, APP, and amyloid-b.
  • the increased levels of phospho-tau and APP immunoreactivity were localized in neurons and neuritic-like plaques.
  • the findings with respectto phospho-tau are consistent with previous studies, which correlated over-expression of AD7c-NTP with phospho-tau- accumulation in Alzheimer's Disease brains and CSF.
  • the findings indicate that increased AD7c-NTP expression precedes phospho-tau accumulation in the course of Alzheimer's Disease associated neurodegeneration, consistent previous observations in brain tissue taken from patients with Alzheimer's Disease.
  • AD7c-NTP Over-expression of AD7c-NTP resulted in increased levels of NOS-3 expression in numerous cortical neurons, as well as in the microvasculature. The distribution of aberrantly increased NOS-3 expression paralleled that of APP. Additional studies in which brains were inoculated with pNOS-3 were done to determine if the increased expression NOS-3 represented a secondary response to AD7c-NTP over-expression. The findings were that NOS-3 over-expression resulted in increased levels of NOS-3, APP, and amyloid-b immunoreactivity, as well as increased p53 and Bax pro-apoptosis gene expression, but had no effect on the levels of AD7c-NTP.
  • the brain expresses reduced levels of cGMP due to increased degradation by phosphodiesterases. Reduced NO signaling through cGMP-dependent pathways results in increased oxidative stress and heightened sensitivity to amyloid-b-toxicity. With regard to AD7c-NTP over-expression, the high levels of NOS-3 and APP likely overwhelm G-protein signaling mechanisms, leading to increased oxidative stress and activation of pro-apoptosis signaling pathways. In the human brain, another important factor contributing to oxidative stress-mediated the activation of the AD7c-NTP-NOS-3-APP-apoptosis cascade with aging and neurodegeneration is progressive impairment of mitochondrial function due to cumulative mitochondrial DNA damage.
  • AD-associated molecular abnormalities including amyloid-b deposition, increased phospho-tau expression, and NOS activation.
  • the data indicate the following scheme in the progression of Alzheimer's Disease (e.g., sporadic Alzheimer's Disease): High levels of AD7c-NTP ⁇ increased NOS-3 expression ⁇ increased APP ⁇ overwhelming of cGMP pathway ⁇ NO-mediated oxidative stress ⁇ phospho-Tau accumulation and activation of pro-apoptosis mechanisms.
  • the scheme indicates that there are multiple points of interfering with the adverse effects of AD7c-NTP over-expression to at least partially rescue of Alzheimer's Disease associated neurodegeneration.

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Claims (2)

  1. Verwendung einer Zusammensetzung, die eine Nucleinsäure, ein Histon und eine amphipathische Verbindung aufweist, zur Herstellung eines Arzneimittels zur Veranlassung einer Langzeit-Genexpression in vivo in einem nicht-menschlichen Säugetier, um dadurch ein nicht-transgenes Tiermodell für Alzheimer-Krankheit zu erzeugen,
    wobei das Arzneimittel zur Verabreichung an Gewebe des zentralen Nervensystems (ZNS) ist,
    wobei die Nucleinsäure ein AD7c-NTP-Polypeptid kodiert,
    und wobei die Genexpression in dem Gewebe nach Verabreichung an das Gewebe mindestens 48 Stunden lang in dem Säugetier nachgewiesen wird.
  2. Zusammensetzung, die eine Nucleinsäure, ein Histon und eine amphipathische Verbindung aufweist, zur Veranlassung einer Langzeit-Expression in vivo in einem nicht-menschlichen Säugetier durch Verabreichung an ZNS-Gewebe , um dadurch ein nicht-menschliches nicht-transgenes Tiermodell für Alzheimer-Krankheit herzustellen,
    wobei die Nucleinsäure ein AD7c-NTP-Polypeptid kodiert.
EP02739383A 2001-06-01 2002-05-28 Hemmung der neurodegeneration Expired - Lifetime EP1414478B1 (de)

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US872968 2001-06-01
US09/872,968 US6770797B2 (en) 2001-06-01 2001-06-01 Non-Transgenic nonhuman model for Alzheimer's Disease using a AD7c-NTP nucleic acid
PCT/US2002/016429 WO2002099036A2 (en) 2001-06-01 2002-05-28 Inhibition of neurodegeneration

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WO2002089841A2 (en) * 2001-05-04 2002-11-14 Nymox Corporation Method of preventing cell death using antibodies to neural thread proteins
JP2005535580A (ja) * 2002-04-26 2005-11-24 イアン， エイ． ファーガソン， 血液脳関門を通したポリペプチドの非侵襲的な送達およびエンドサイトーシスリガンドの生体内選択
AU2003233120B2 (en) * 2002-04-26 2008-08-21 Ferguson, Ian Andrew Non-invasive delivery of polypeptides through the blood-brain barrier, and in vivo selection of endocytotic ligands
US7544771B2 (en) * 2005-02-23 2009-06-09 Nymox Corporation Protein and its use in diagnosing Alzheimer's disease
EP2381774A4 (de) * 2008-12-23 2012-07-18 Salk Inst For Biological Studi Verfahren zur behandlung von neurodegenerativen erkrankungen
WO2011084598A1 (en) * 2010-01-08 2011-07-14 Oncohealth Corporation High throughput cell-based hpv immunoassays for diagnosis and screening of hpv-associated cancers

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5948634A (en) 1988-12-21 1999-09-07 The General Hospital Coporation Neural thread protein gene expression and detection of alzheimer's disease
US5800390A (en) 1991-05-24 1998-09-01 Sumitomo Pharmaceuticals Company, Limited Equipment for intracerebral administration of preparations
JP3513859B2 (ja) 1993-04-20 2004-03-31 ザ・ジェネラル・ホスピタル・コーポレーション 神経糸状タンパク質遺伝子発現およびアルツハイマー病の検出
US5744335A (en) 1995-09-19 1998-04-28 Mirus Corporation Process of transfecting a cell with a polynucleotide mixed with an amphipathic compound and a DNA-binding protein
US5863898A (en) 1996-10-29 1999-01-26 Incyte Pharmaceuticals, Inc. Human lim proteins
JP3758792B2 (ja) 1997-02-25 2006-03-22 三菱重工業株式会社 ガスタービン動翼のプラットフォーム冷却機構
EP0975651A4 (de) 1997-02-26 2005-03-09 Gen Hospital Corp Transgene tiere und zellinien zur erkennung von zur behandlung oder vorbeugung der alzheimerschen krankheit wirksamen medikamente
EP1006794B1 (de) 1997-03-12 2007-11-28 Robert W. Esmond Verfahren zur behandlung oder prävention der alzheimerischen krankheit

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JP2005516886A (ja) 2005-06-09
CA2448863A1 (en) 2002-12-12
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CA2448863C (en) 2010-05-04
AU2002312033B2 (en) 2007-10-18
ATE473009T1 (de) 2010-07-15
EP1414478A4 (de) 2007-03-21
US6770797B2 (en) 2004-08-03
US20050090441A1 (en) 2005-04-28
US7319094B2 (en) 2008-01-15
WO2002099036A2 (en) 2002-12-12
WO2002099036A3 (en) 2004-01-08
US20030050262A1 (en) 2003-03-13

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